The epidermal growth factor receptor (EGFR) tyrosine kinase pathway has been demonstrated to be a key molecular target for cancer therapeutics including TKIs such as Iressa (gefinitib) and Tarceva (erlotinib). These compounds are small molecule ATP mimetics, selectively binding to the intracellular kinase of EGFR, that have recently received FDA approval for treatment of non-small cell lung carcinoma (NSCLC). An ever growing body of data indicates that the presence of mutations in EGFR play an important role in response to TKI therapy and drug resistance. There are a subset of patients (~10%) who have a remarkable response to Iressa and Tarceva that have now been correlated to several specific activating EGFR mutations arising within the tumor. A second mutation has been identified in relapsed patients who were initially drug responsive and more recently this mutation has also been found in a family with a genetic predisposition for developing lung cancer. Recent studies from our lab and others suggest an emerging new paradigm for modulation of downstream signaling. These studies show that autophosphorylation of RTKs occurs in a specific sequential order, creating a temporal and dynamic phosphorylation pattern of different intermediate sets of phosphotyrosines. Accordingly, these discrete sets of phosphotyrosines can orchestrate the dynamic recruitment of specific downstream signaling partners in a temporal fashion that may well play an important role in the proper regulation of protein signaling. The underlying molecular mechanisms of the oncogenic activating and resistance mutations in EGFR and differential sensitivity to Iressa and Tarceva are not understood. Our preliminary data suggest that the underlying causes may be linked to alterations in nucleotide affinity for the receptor, kinase catalytic activity, and/or alterations in phosphorylation patterns that result in differential protein signaling. The studies outlined in this application will use a combination of in vitro biochemical studies and complementary cellular studies to identify specific molecular alterations that may distinguish wild type (WT) and oncogenic forms of EGFR harboring Iressa/Tarceva sensitive activating mutations as well as mutations associated with drug resistance. The overall objectives are to define the molecular mechanism of protein signaling in the EGFR pathway under normal and aberrant oncogenic processes and provide an understanding for the modulating effects of Iressa/Tarceva. This information will ultimately aid in the design of more potent and selective compounds for precisely targeted cancer therapy. Growth factors bind to their corresponding receptors located on the surface of cells and give signals for cell growth. In a cancer cell, the signals for cell growth have aberrant signaling and growth and proliferation have become out of control. The newest, most promising cancer therapies such as Iressa and Tarceva, targets these uncontrolled signaling pathways in cancer cells. Some patients with non- small cell lung carcinoma having particular mutations in their growth factor receptors respond very well to these drugs, however the molecular events are not understood. The studies described in this application are designed help unravel these molecular details to allow us to differentiate cancer cells from normal cells and assess the usefulness of current and new cancer therapies.